Project Summary Tobacco smoking remains the leading cause of premature death in the United States. While quitting smoking is a major obstacle, avoiding relapse is an even greater challenge. Stress is a well-established factor driving relapse to smoking in humans and nicotine seeking in animal models. Stress activates the noradrenergic system, and specific noradrenergic projections from lateral tegmental nuclei in the brainstem, including the nucleus tractus solitarius (NTS), are critical for stress-induced reinstatement of drug seeking. Modulating noradrenergic signaling, for example with guanfacine, an ?2 adrenergic receptor agonist, is a promising therapeutic target to reduce relapse. Importantly, evidence suggests that sex interacts with stress to enhance not only risk for relapse but also response to treatments targeting the stress response. Our lab has shown sex differences in response to guanfacine as sex-specific patterns of c-fos, a marker of neuronal activity, in the basolateral amygdala. However, the neurobiological underpinnings of sex differences in how noradrenergic agents affect stress-induced reinstatement of nicotine seeking are unclear. We propose to use pharmacologic and chemogenetic techniques to investigate behavioral sex differences in stress-induced reinstatement of nicotine seeking and a circuit-level mechanism for these sex differences in NTS noradrenergic projections. Aim 1 will test the hypothesis that guanfacine attenuates stress-induced reinstatement of nicotine seeking to a greater degree in female than male mice. I will use a conditioned place preference model of nicotine seeking to assess stress-induced reinstatement and the effects of systemic guanfacine on this behavior. Aim 2 will test the hypothesis that the NTS noradrenergic neurons are a neurobiological substrate contributing to sex differences in stress-induced reinstatement of nicotine seeking. I will use a combination of chemogenetics and transgenic TH-Cre mice to selectively target NTS noradrenergic projections. Experiment 2a will use inhibitory DREADDs, and Experiment 2b will use excitatory DREADDs to decrease and increase, respectively, NTS noradrenergic activity and test the effects on reinstatement of nicotine seeking in male and female mice. These experiments will further our understanding of how sex and stress interact to affect relapse to nicotine seeking and how activity in specific noradrenergic circuits contributes to this interaction. Understanding these sex differences and their neurobiological underpinnings will ultimately contribute to the development of targeted smoking cessation therapies to help more smokers maintain abstinence.